A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant.

A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-ß-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR.

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects.

Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and ß) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/ß)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/ß) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/ß)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90ß/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/ß)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.

Positron emission tomography reporter genes and reporter probes: gene and cell therapy applications.

Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.

Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe.

Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-ß-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.

Lymphoid-tissue-specific homing of bone-marrow-derived dendritic cells.

Because of their potent immunoregulatory capacity, dendritic cells (DCs) have been exploited as therapeutic tools to boost immune responses against tumors or pathogens, or dampen autoimmune or allergic responses. Murine bone marrow-derived DCs (BM-DCs) are the closest known equivalent of the blood monocyte-derived DCs that have been used for human therapy. Current imaging methods have proven unable to properly address the migration of injected DCs to small and deep tissues in mice and humans. This study presents the first extensive analysis of BM-DC homing to lymph nodes (and other selected tissues) after intravenous and intraperitoneal inoculation. After intravenous delivery, DCs accumulated in the spleen, and preferentially in the pancreatic and lung-draining lymph nodes. In contrast, DCs injected intraperitoneally were found predominantly in peritoneal lymph nodes (pancreatic in particular), and in omentum-associated lymphoid tissue. This uneven distribution of BM-DCs, independent of the mouse strain and also observed within pancreatic lymph nodes, resulted in the uneven induction of immune response in different lymphoid tissues. These data have important implications for the design of systemic cellular therapy with DCs, and in particular underlie a previously unsuspected potential for specific treatment of diseases such as autoimmune diabetes and pancreatic cancer.

Novel strategy for a cocktail 18F-fluoride and 18F-FDG PET/CT scan for evaluation of malignancy: results of the pilot-phase study.

UNLABELLED: (18)F-FDG PET/CT is used for detecting cancer and monitoring cancer response to therapy. However, because of the variable rates of glucose metabolism, not all cancers are identified reliably. Sodium (18)F was previously used for bone imaging and can be used as a PET/CT skeletal tracer. The combined administration of (18)F and (18)F-FDG in a single PET/CT study for cancer detection has not been reported to date. METHODS: This is a prospective pilot study (November 2007-November 2008) of 14 patients with proven malignancy (6 sarcoma, 3 prostate cancer, 2 breast cancer, 1 colon cancer, 1 lung cancer, and 1 malignant paraganglioma) who underwent separate (18)F PET/CT and (18)F-FDG PET/CT and combined (18)F/(18)F-FDG PET/CT scans for the evaluation of malignancy (a total of 3 scans each). There were 11 men and 3 women (age range, 19-75 y; average, 50.4 y). RESULTS: Interpretation of the combined (18)F/(18)F-FDG PET/CT scans compared favorably with that of the (18)F-FDG PET/CT (no lesions missed) and the (18)F PET/CT scans (only 1 skull lesion seen on an (18)F PET/CT scan was missed on the corresponding combined scan). Through image processing, the combined (18)F/(18)F-FDG scan yielded results for bone radiotracer uptake comparable to those of the (18)F PET/CT scan performed separately. CONCLUSION: Our pilot-phase prospective trial demonstrates that the combined (18)F/(18)F-FDG administration followed by a single PET/CT scan is feasible for cancer detection. This combined method opens the possibility for improved patient care and reduction in health care costs.

A novel method for direct site-specific radiolabeling of peptides using [18F]FDG.

We have used the well-accepted and easily available 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) positron emission tomography (PET) tracer as a prosthetic group for synthesis of (18)F-labeled peptides. We herein report the synthesis of [(18)F]FDG-RGD ((18)F labeled linear RGD) and [(18)F]FDG-cyclo(RGD(D)YK) ((18)F labeled cyclic RGD) as examples of the use of [(18)F]FDG. We have successfully prepared [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) in 27.5% and 41% radiochemical yields (decay corrected) respectively. The receptor binding affinity study of FDG-cyclo(RGD(D)YK) for integrin alpha(v)beta(3), using alpha(v)beta(3) positive U87MG cells confirmed a competitive displacement with (125)I-echistatin as a radioligand. The IC(50) value for FDG-cyclo(RGD(D)YK) was determined to be 0.67 +/- 0.19 muM. High-contrast small animal PET images with relatively moderate tumor uptake were observed for [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) as PET probes in xenograft models expressing alpha(v)beta(3) integrin. In conclusion, we have successfully used [(18)F]FDG as a prosthetic group to prepare (18)F]FDG-RGD and [(18)F]FDG-cyclic[RGD(D)YK] based on a simple one-step radiosynthesis. The one-step radiosynthesis methodology consists of chemoselective oxime formation between an aminooxy-functionalized peptide and [(18)F]FDG. The results have implications for radiolabeling of other macromolecules and would lead to a very simple strategy for routine preclinical and clinical use.

Noninvasive detection of therapeutic cytolytic T cells with 18F-FHBG PET in a patient with glioma.

BACKGROUND: A 57-year-old man had been diagnosed with grade IV glioblastoma multiforme and was enrolled in a trial of adoptive cellular immunotherapy. The trial involved infusion of ex vivo expanded autologous cytolytic CD8+ T cells (CTLs), genetically engineered to express the interleukin 13 zetakine gene (which encodes a receptor protein that targets these T cells to tumor cells) and the herpes simplex virus 1 thymidine kinase (HSV1 tk) suicide gene, and PET imaging reporter gene. INVESTIGATIONS: MRI, whole-body and brain PET scan with (18)F-radiolabelled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) to detect CTLs that express HSV1 tk, and safety monitoring after injection of (18)F-FHBG. DIAGNOSIS: MRI detected grade III-IV glioblastoma multiforme plus two tumors recurrences that developed after resection of the initial tumor. MANAGEMENT: Surgical resection of primary glioblastoma tumor, enrollment in CTL therapy trial, reresection of glioma recurrences, infusion of approximately 1 x 10(9) CTLs into the site of tumor reresection, and (18)F-FHBG PET scan to detect infused CTLs.

Tissue-targeted therapy of autoimmune diabetes using dendritic cells transduced to express IL-4 in NOD mice.

A deficit in IL-4 production has been previously reported in both diabetic human patients and non-obese diabetic (NOD) mice. In addition, re-introducing IL-4 into NOD mice systemically, or as a transgene, led to a beneficial outcome in most studies. Here, we show that prediabetic, 12-week old female NOD mice have a deficit in IL-4 expression in the pancreatic lymph nodes (PLN) compared to age-matched diabetes-resistant NOD.B10 mice. By bioluminescence imaging, we demonstrated that the PLN was preferentially targeted by bone marrow-derived dendritic cells (DCs) following intravenous (IV) administration. Following IV injection of DCs transduced to express IL-4 (DC/IL-4) into 12-week old NOD mice, it was possible to significantly delay or prevent the onset of hyperglycemia. We then focused on the PLN to monitor, by microarray analysis, changes in gene expression induced by DC/IL-4 and observed a rapid normalization of the expression of many genes, that were otherwise under-expressed compared to NOD.B10 PLN. The protective effect of DC/IL-4 required both MHC and IL-4 expression by the DCs. Thus, adoptive cellular therapy, using DCs modified to express IL-4, offers an effective, tissue-targeted cellular therapy to prevent diabetes in NOD mice at an advanced stage of pre-diabetes, and may offer a safe approach to consider for treatment of high risk human pre-diabetic patients.

Studying the biodistribution of positron emission tomography reporter probes in mice.

Positron emission tomography (PET) reporter probes (PRPs) are used to detect PET reporter gene (PRG) expression in living subjects. This article details protocols for analyzing the biodistribution of a PRP used to detect herpes simplex virus 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk PRG expression. However, the methods described are generalizable to other beta- or gamma/positron-emitting probes. Accumulation of PRPs in animal tissues can be determined by counting PRP activity of isolated tissues, whereas digital whole-body autoradiography (DWBA) provides high-resolution images of PRP biodistribution in 5- to 45-microm tissue slices of killed research animals at a single time point. Biodistribution assay results may be obtained in less than a week after beginning the assay, and DWBA image acquisitions can take up to 3 months depending on the probe's radioisotope.

Multimodality imaging of T-cell hybridoma trafficking in collagen-induced arthritic mice: image-based estimation of the number of cells accumulating in mouse paws.

Appropriate targeting of therapeutic cells is essential in adoptive cellular gene therapy (ACGT). Imaging cell trafficking in animal models and patients will guide development of ACGT protocols. Collagen type II (C-II)-specific T cell hybridomas are transduced with a lentivirus carrying a triple fusion reporter gene (TFR) construct consisting of a fluorescent reporter gene (RG), a bioluminescent RG (hRluc), and a positron emission tomography (PET) RG. Collagen-induced arthritic (CIA) mice are scanned with a bioluminescence imaging camera before and after implantation of various known cell quantities in their paws. Linear regression analysis yields equations relating two parameters of image signal intensity in mice paws to the quantity of hRluc expressing cells in the paws. Afterward, trafficking of intravenously injected cells is studied by quantitative analysis of bioluminescence images. Comparison of the average cell numbers does not demonstrate consistently higher accumulation of T-cell hybridomas in the paws with higher inflammation scores, and injecting more cells does not cause increased accumulation. MicroPET images illustrate above background signal in the inflamed paws and chest areas of CIA mice. The procedures described in this study can be used to derive equations for cells expressing other bioluminescent RGs and in other animal models.

Preclinical safety evaluation of 18F-FHBG: a PET reporter probe for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk's expression.

UNLABELLED: 9-(4-(18)F-Fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG) is a sensitive and specific PET reporter probe for imaging the PET reporter genes, herpes simplex 1 thymidine kinase (HSV1-tk) and its mutant HSV1-sr39tk. (18)F-FHBG has suitable pharmacokinetics and dosimetry for clinical applications and imaging of HSV1-TK has been demonstrated in the livers of hepatocellular cancer patients. METHODS: Male and female Sprague-Dawley rats and New Zealand White rabbits were divided into equal groups receiving either 14 microg/kg cold FHBG or carrier solution, for a 14-d acute toxicity assessment. We monitored body weight, food and water consumption, body temperature, cardiovascular electrical and functional indices, respiratory performance and oxygen saturation, comprehensive blood chemistry, complete blood count (CBC), and urinalysis. We conducted daily cage-side examinations for the detection of any clinical abnormalities. Tissues of the animals that were euthanized and necropsied on day 14 were prepared for histopathologic examination. RESULTS: No significant differences in cardiovascular and respiratory parameters, food consumption, body weight, urine components, or clinical signs attributable to test article toxicity were observed between the treatment and control groups. Any differences noted in the blood chemistry and CBC parameters were deemed to be incidental findings unrelated to the administration of the FHBG. CONCLUSION: Acute toxicity evaluation of FHBG at 100 times the expected human dose does not indicate harm to organ function or tissues. The Food and Drug Administration has approved FHBG as an Investigational New Drug.

Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro.

The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.

PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG.

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and to image intracellular molecular events and cell trafficking in living subjects. The expression of these PRGs can be imaged using 18F- or 124I-radiolabeled acycloguanosine or pyrimidine analog PET reporter probes (PRPs). This protocol describes the procedures for imaging HSV1-tk or HSV1-sr39tk PRG expression in living subjects with the acycloguanosine analog 9-4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG). [18F]FHBG is a high-affinity substrate for the HSV1-sr39TK enzyme with relatively low affinity for mammalian TK enzymes, resulting in improved detection sensitivity. Furthermore, [18F]FHBG is approved by the US Food and Drug Administration as an investigational new imaging agent and has been shown to detect HSV1-tk transgene expression in the liver tumors of patients. MicroPET imaging of each small animal can be completed in approximately 1.5 h, and each patient imaging session takes approximately 3 h.

Imaging progress of herpes simplex virus type 1 thymidine kinase suicide gene therapy in living subjects with positron emission tomography.

Molecular imaging of a suicide transgene's expression will aid the development of efficient and precise targeting strategies, and imaging for cancer cell viability may assess therapeutic efficacy. We used the PET reporter probe, 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)guanine ([18F]FHBG) to monitor the expression of a mutant Herpes Simplex Virus 1 thymidine kinase (HSV1-sr39tk) in C6 glioma tumors implanted subcutaneously in nude mice that were repetitively being treated with the pro-drug Ganciclovir (GCV). [18F]-Fluorodeoxyglucose ([18F]FDG), a metabolic tracer, was used to assess tumor cell viability and therapeutic efficacy. C6 glioma tumors stably expressing the HSV1-sr39tk gene (C6sr39) accumulated [18F]FHBG prior to GCV treatment. Significant declines in C6sr39 tumor volumes and [18F]FHBG and [18F]FDG accumulation were observed following 2 weeks of GCV treatment. However, 3 weeks after halting GCV treatment, the tumors re-grew and [18F]FDG accumulation increased significantly; in contrast, tumor [18F]FHBG concentrations remained at background levels. Therefore, [18F]FHBG can be used to detect tumors expressing HSV1-sr39tk, susceptible to regression in response to GCV exposure, and the effectiveness of GCV therapy in eradicating HSV1-sr39tk-expressing cells can be monitored by [18F]FHBG scanning. [18F]FHBG and [18F]FDG imaging data indicate that exposure of C6sr39 tumors to GCV causes the elimination of [18F]FHBG-accumulating C6sr39 cells and selects for re-growth of tumors unable to accumulate [18F]FHBG.